Process for producing acrylamide using a microbial catalyst having been wahed with aqueous acrylic acid solution

ABSTRACT

Provided is a process for producing acrylamide with good storage stability and improved acrylamide polymer physical properties using a microbial catalyst. A microbial catalyst having catalytic activity to convert from acrylonitrile to acrylamide is washed with an aqueous acrylic acid solution, and then the washed microbial catalyst is used for the conversion reaction, so that the production of the above acrylamide is achieved.

TECHNICAL FIELD

[0001] The present invention relates to a process for producingacrylamide from acrylonitrile by the action of a microorganism-derivedenzyme, nitrilehydratase. Acrylamide is used in a variety of fields asan industrially important substance. For example, acrylamide polymersare widely used in applications such as a coagulant for wastewatertreatment, a paper strong agent, an oil collecting agent and the like.

BACKGROUND ART

[0002] Acrylamide has been conventionally produced industrially byhydrating acrylonitrile corresponding thereto using copper in thereduced state as a catalyst. Recently, a method using a microbialcatalyst instead of a copper catalyst has been developed, and a part ofthis method is in actual use. A biocatalytic method using a microbialcatalyst or the like has promise as an industrial production method,because the method has moderate reaction conditions and yields almost noby-product, so that an extremely simple process can be designed for thismethod. Thus, many microorganisms having an enzyme (enzyme name:nitrilehydratase) capable of catalyzing (converting) acrylonitrile intoacrylamide by hydration have been found so far.

[0003] Examples of these microorganisms include microbial strainsbelonging to the genera Bacillus, Bacteridium, Micrococcus,Brevibacterium [see JP Patent Publication (Kokoku) No. 62-21519 B (1987)for the above microorganisms], Corynebacterium and Nocardia [see JPPatent Publication (Kokoku) No. 56-17918 B (1981) for the abovemicroorganisms], Pseudomonas [see JP Patent Publication (Kokoku) No.59-37951 B (1984)], Rhodococcus and Microbacterium [see JP PatentPublication (Kokoku) No. 4-4873 B (1992) for the above microorganisms],Rhodococcus rhodochrous [see JP Patent Publication (Kokoku) No. 6-55148B (1994)], and Rhodococcus [see JP Patent Publication (Kokoku) No.7-40948 B (1995)].

[0004] Examples of a process for producing acrylamide using the abovemicroorganism as a microbial catalyst include those of JP PatentPublication (Kokai) Nos. 11-123098 A (1999) and 7-265091 A (1995), andJP Patent Publication (Kokoku) No. 56-38118 B (1981). An example of thereaction method is that of JP Patent Publication (Kokai) No. 11-89575 A(1999).

[0005] Further, a variety of studies have been conducted for improvingenzyme activity or suppressing a decrease (deactivation of) in enzymeactivity during reaction. Examples of such a study include a processwhich involves performing reaction at a low temperature, 15° C. belowfreezing point [see JP Patent Publication (Kokoku) No. 56-38118 B(1981)], a process which involves sequentially supplying a substrate ata low concentration from multiple supply openings [see JP PatentPublication (Kokoku) No. 57-1234 B (1982)], a process which involvestreating microorganisms or the treated product thereof with an organicsolvent [see JP Patent Publication (Kokai) No. 5-308980 A (1993)] aprocess which involves performing reaction under the presence of higherunsaturated fatty acid [see JP Patent Publication (Kokai) No. 7-265090 A(1995)], and a process which involves subjecting microbial cells tocross-linking treatment with glutaraldehyde or the like [see JP PatentPublication (Kokai) Nos. 7-265091 A (1995) and 8-154691 A (1996)].

[0006] In the meantime, for washing a microbial catalyst, the generallyknown methods involve washing using a physiological saline, a buffersuch as an aqueous solution of phosphate or Tris hydrochroride tosuppress a decrease in enzyme activity. However, there is no report onthe washing of a microbial catalyst wherein the effects of washcomponents on the physical properties of acrylamide polymers and thestorage stability of monomers have been considered.

[0007] As described above, a process for producing acrylamide using amicrobial catalyst has promise as an industrial production process,because the process employs moderate reaction conditions and yieldsalmost no by-product, so that no purification is required and anextremely simple process may be designed.

[0008] Although the above production processes yield no by-product uponenzyme reaction, they have a drawback such that when a microbialcatalyst to be used is washed, contamination of impurities derived fromthe wash affects the physical properties of acrylamide polymers and thestorage stability of acrylamide monomers. To address the problem,purification such as crystallization, ion exchange, or distillation canbe performed. With these purification processes, however, an outstandingcharacteristic of a production process using a microbial catalyst, thatis, to yield almost no by-product upon reaction, cannot be utilized.Moreover, the use of these processes is also unfavorable in terms ofenergy and environmental problems.

SUMMARY OF THE INVENTION

[0009] As a result of intensive studies to address the above problems,we have completed the present invention by finding that acrylamide withimproved physical properties of acrylamide polymers and improved storagestability of acrylamide monomers can be produced by using a microbialcatalyst washed with an aqueous acrylic acid solution in a process forproducing acrylamide from acrylonitrile using a microbial catalysthaving a microorganism-derived enzyme, nitrilehydratase.

[0010] In other words, the present invention is a process for producingacrylamide using a microbial catalyst that converts acrylonitrile toacrylamide, which uses the microbial catalyst having been washed with anaqueous acrylic acid solution.

[0011] The microbial catalyst that can be used in the present inventionmay be any catalyst as long as it is prepared from microorganisms havingcatalytic activity (nitrilehydratase activity) to convert acrylonitrileto acrylamide. Preferred examples of such microbial species includethose belonging to the genus Bacillus, genus Bacteridium, genusMicrococcus, genus Brevibacterium, genus Corynebacterium, genusNocardia, genus Pseudomonas, genus Microbacterium, genus Rhodococcus,genus Achromobacter, and genus Pseudonocardia. One of or a combinationof these microorganisms can be used.

[0012] Further, a transformant that may be used herein is prepared byobtaining a nitrilehydratase gene derived from the above microorganism,and then introducing the gene directly, or the artificially improvedgene, into a freely chosen host.

[0013] Preferred examples of the above transformant include Escherichiacoli MT10770 (FERM P-14756) (JP Patent Publication (Kokai) No. 8-266277A (1996)) that has been transformed with nitrilehydratase of the genusAchromobacter, Escherichia coli MT10822 ((FERM BP-5785) (JP PatentPublication (Kokai) No. 9-275978 A (1997)) that has been transformedwith nitrilehydratase of the genus Pseudonocardia, or microorganismstransformed with nitrile-hydratase (JP Patent Publication (Kokai) No.4-211379 A (1992) of the genus Rhodococcus rhodochrous.

[0014] The above microorganisms can be cultured by any method that isappropriate for a given microbial species.

[0015] In the present invention, the microbial catalyst that is preparedfrom microorganisms refers to a culture solution obtained by culturingmicroorganisms, cells obtained by a harvesting process or the like,cells disrupted by ultrasonication or the like, or those prepared aftercell disruption including a crude enzyme, a partially-purified enzyme ora purified enzyme. If necessary, these microbial catalysts may beimmobilized on carriers such as polyacrylamide gel, alginate,carrageenan or ion exchange resin. A mode to use the microbial catalystmay be appropriately selected depending on enzyme stability, productionscale and the like.

[0016] The term “washing” in the present invention refers to the washingof microbial cells that have been cultured and/or microbial catalysts tobe used in a reaction. Thus, both microbial cells that have beencultured and microbial catalysts to be used in a reaction may be washedwith acrylic acid, or only the microbial catalysts to be used in areaction may be washed with acrylic acid. For example, a microbialcatalyst to be used in a reaction may be washed once with water, abuffer or the like, and then washed with acrylic acid before thereaction. Microbial catalysts may be washed with acrylic acidimmediately before the reaction.

[0017] Further, any washing method can be employed. Examples of such amethod that can be illustrated herein include a method which involvesrepeated washing and centrifugation, and a washing method using a hollowfiber membrane. Further, immobilized microbial catalysts can be washedby repeating agitation and precipitation of the immobilized catalysts ina wash and the removal of supernatant.

[0018] Any washing method and any number of washing can be appropriatelyset in consideration of washing efficiency, enzyme stability and thelike.

[0019] The concentration of acrylic acid to be used for washing ispreferably between 0.01% by mass and 10% by mass in an aqueous acrylicacid solution. More preferably, the concentration is between 0.05% bymass and 1% by mass, and most preferably is 0.1% by mass.

[0020] When the concentration of acrylic acid is 0.01% by mass or less,the duration of washing and the number of washing increase, so as tomake the procedures complex. Furthermore, such increased number ofwashing cause cell disruption during washing, collapsed immobilizedcells and the like. 10% by mass or more of the concentration isunfavorable because it causes a decrease in enzyme activity, and is alsounfavorable economically.

[0021] The pH of an aqueous acrylic acid solution is adjusted usingsodium hydroxide, ammonia or the like. Preferably the pH of the solutionused herein is adjusted to be between 5 and 11, more preferably between6 and 10, and most preferably to be 7.

[0022] Microbial catalysts prepared as described above can be used asmicrobial catalysts in a state of suspension or dispersal in an aqueousacrylic acid solution, or in a state of being subjected to solid-liquidseparation.

BEST MODE OF CARRYING OUT THE INVENTION

[0023] The present invention will be described more specifically by thefollowing examples. These examples are not intended to limit the scopeof the present invention.

EXAMPLE 1 (1) Process for Preparing Cultured and Washed Microbial Cells

[0024] Rhodococcus rhodochrous J-1 (FERM BP-1478) havingnitrilehydratase activity (JP Patent Publication (Kokoku) No. 6-55148 B(1994)) was cultured in a medium (pH 7.0) containing 2% by mass ofglucose, 1% by mass of urea, 0.5% by mass of peptone, 0.3% by mass ofyeast extract, and 0.05% by mass of cobalt chloride in a 30 L jarfermenter (Takasugi Seisakusho) at 30° C. for 60 hours aerobically.

[0025] 20 liters of the solution cultured as described above werefiltered by circulation through a cross flow type hollow fiber membranemodule. The cultured cells were washed by sequentially supplying 0.7% bymass of phosphate buffer (pH 7.0) in a volume corresponding to thevolume of the filtrate to the culture solution, thereby obtaining washedmicrobial cells.

(2) Preparation of Microorganism-Immobilized Carriers

[0026] To 500 g of the washed cell suspension (20% by mass whenconverted into dry cell weight) obtained in (1), 500 g of a monomermixture solution containing acrylamide, methylene bisacrylamide and2-dimethylaminopropylmethacrylamide with a concentration of 20%, 2% and2% by mass, respectively, was added, so as to perform suspension well.5% by mass (2 g) of ammonium persulfate and 50% by mass (2 g) ofN,N,N,N-tetramethylethylenediamine were added to the suspension forpolymerization and gelatinization. The product was cut into anapproximately 1-mm cube, thereby obtaining microorganism-immobilizedcarriers.

[0027] The microorganism-immobilized carrier obtained by the abovemethod was subjected to 20 cycles of a procedure, each cycle consistingof the following steps: (1) suspension and agitation in a 0.1% by massaqueous sodium acrylate solution (pH 7.0), (2) still standing andprecipitation, and (3) disposal of supernatant.

(3) Amidation Reaction

[0028] 3200g of a 0.2 g/L aqueous sodium acrylate solution was put in aseparable flask with an internal volume of 5 liters. To the aqueousacrylic acid solution, 3 g of the immobilized microorganisms prepared in(2) was added. The solution was agitated while maintaining pH 7.0 and atemperature of 20° C.

[0029] To this solution, acrylonitrile was sequentially fed for keepingthe concentration of acrylonitrile at 2% by mass, and then anaccumulation reaction was performed until the acrylamide concentrationbecame 50% by mass.

[0030] After the end of the reaction, the solution was filtered througha membrane filter with an aperture of 0.45 μm, so as to remove thecatalyst.

(4) Method for Evaluating the Physical Properties of Polymer

[0031] The 20% by mass of acrylamide obtained in Example 1(3) wasdissolved in 80% by mass of water. After the pH was adjusted to 8.0, thesolution was transferred into a Dewar flask, and then the air within thesystem was replaced by nitrogen. Then, 0.0004% of ammonium persulfate,0.0004% of iron sulfate, and 0.01% of 4,4′-azobis-(4-cyanovaleric acid)were added to perform polymerization. The thus obtained water-containinggelatinous polymers were shredded into particles with a diameter ofseveral mm using a meat mincer. The particles were then dried at 80° C.for 10 hours, and then disrupted to have a particle size of 2 mm or lessusing a Wiley grinder, thereby obtaining polymer powder.

[0032] The thus obtained polymer powder was prepared to have aconcentration of 0.2% with 500 g of water. The solution was agitated atroom temperature for 4 hours and then dissolved. The Brookfieldviscosity (type B viscometer, the number of rotation of a rotor: 30 rpm,and Rotor No. 1) was then measured. Then, the solution was filteredthrough a 80-mesh woven metal wire, and then the mass of insolublematters that had remained on the wire after washing with water wasmeasured.

(5) Method for Evaluating Monomer Storage Stability

[0033] 50 g of the 50% acrylamide monomer aqueous solution obtained inExample 1 (3) and iron test pieces were put in a 100 mlpolyethylene-made bottle, and then the bottle was closed with a cap toavoid evaporation. This bottle was stored in a high temperature box at50° C., and then the stability was determined based on the presence orabsence of polymerized products.

[0034] The results of Example 1 (4) and (5) are shown in Table 1 andTable 2.

COMPARATIVE EXAMPLE 1

[0035] Comparative example 1 was performed similarly to Example 1 exceptfor using a 0.7% phosphate buffer (pH 7.0) instead of 0.1% by mass ofaqueous sodium acrylate solution (pH 7.0) in the step of preparingmicroorganism-immobilized carriers in Example 1 (2). The results areshown in Table 1 and Table 2. TABLE 1 Physical properties of polymerTime for Insoluble polymerization Viscosity matter [min.] [mPa · s] [g]Example 1 17 44 0 Comparative 74 150 290 example 1

[0036] TABLE 2 Storage stability Days of storage [day] Example 1 >10Comparative <1 example 1

[0037] All publications, patents and patent applications cited hereinare incorporated herein by reference in their entirety.

Industrial Applicability

[0038] As described in detail above, the use of a microbial catalystthat has been washed with acrylic acid when acrylamide is produced usinga microbial catalyst makes it possible to obtain acrylamide with highstorage stability and good quality.

1. A process for producing acrylamide using a microorganism havingnitrilehydratase, which uses a microbial catalyst having been washedwith an aqueous acrylic acid solution.
 2. The process for producingacrylamide according to claim 1, wherein the microbial catalyst isprepared from at least one microorganism selected from the groupconsisting of the genus Bacillus, genus Bacteridium, genus Micrococcus,genus Brevibacterium, genus Corynebacterium, genus Nocardia, genusPseudomonas, genus Microbacterium, genus Rhodococcus, genusAchromobacter, and genus Pseudonocardia.
 3. The process for producingacrylamide according to claim 1 or 2, wherein the concentration ofacrylic acid in the aqueous acrylic acid solution is between 0.01% bymass and 10% by mass.
 4. The process for producing acrylamide accordingto any one of claims 1 to 3, wherein the pH of the aqueous acrylic acidsolution is between 5 and 11.